MIKROBIOLOGI INDUSTRI

TK141241

Taksonomi Mikroorganisme

Dr. Eng. R. Darmawan

TERMINOLOGY

Taksonomi
Ilmu pengetahuan yang berkaitan dengan klasifikasi, nomenklatur
(penamaan), dan identifikasi organisme ke dalam kelompok/ kategori (taksa)

Klasifikasi
Pengelompokkan organisme menjadi taksa (tunggal : takson) tertentu
berdasarkan karakteristik yang sama dan bukan disusun atas order hierarki

Nomenklatur
Penamaan satuan-satuan yang dicirikan dan dibatasi oleh klasifikasi

Identifikasi
Penggunaan kriteria yang ditetapkan untuk klasifikasi mikroorganisme
dengan membandingkan ciri yang ada pada satuan yang belum diketahui
dengan satuan-satuan yang sudah dikenal.
TERMINOLOGY
Phylogenetic Classification System:
Groups reflect genetic similarity and evolutionary relatedness

Phenetic Classification System:

Groups do not necessarily reflect genetic similarity or evolutionary
relatedness. Instead, groups are based on convenient, observable
characteristics.
TAXONOMY HISTORY

*1700s 2 kingdoms: plant and animal

*1800s 3 kingdoms: plant, animal, and protista
*1950-1990s 5 kingdoms: plant, animal, protista, fungi,
monera
*Present: 5 kingdoms: monera (eubacteria +
archaebacteria), protista, animal, plant, fungi
*Taxonomy consists of 3 parts
*Classification
*Nomenclature
*Identification

(1701-1778)
TAXONOMY HISTORY
A Swedish naturalist named Carolus Linnaeus is considered the 'Father of
Taxonomy‘ since 1700s
His two most important contributions to taxonomy were:
•A hierarchical classification system
•The system of binomial nomenclature

He proposed that there were three broad groups, called kingdoms, into
which the whole of nature could fit. Two kingdoms were animals, plants.

Binomial nomenclature meant naming species in

2 words : genus , followed
by species.

(1701-1778)
TAXONOMY HISTORY (2 KINGDOM)
•The two kingdom classification system was given by Carlous Linaaeus in
1758.

He then divided each kingdom into classes and later grouped the classes
into phyla for animals and divisions for plants
TAXONOMY HISTORY (3 KINGDOM)
TAXONOMY HISTORY (4 KINGDOM)
The development of optic and electronic microscopy showed
important differences in cells, mainly according to the presence or
absence of distinct nucleus, leading Édouard Chatton to distinguish
organisms in prokaryotes (without a distinct nucleus) and eukaryotes (with
a distinct nucleus) in a paper from 1925.

Based on it, Copeland proposed a four-kingdom system, moving

prokaryotic organisms, bacteria and “blue-green algae”, into the kingdom
Monera.
TAXONOMY HISTORY (5 KINGDOM)
The position of fungi was not well established, oscillating between
kingdoms Protista and Plantae.
So, in 1969, Robert Whittaker proposed a fifth kingdom to include
them, the called Kingdom Fungi.
TAXONOMY HISTORY

Year System Given by Basis

(1753) 2 Kingdom Linnaeus Cell wall

(1865) 3 Kingdom Ernst Haeckel Cellularity level

After 1925 4 kingdom Copeland Compartmentalizati

on of cell organelles
(1969) 5 Kingdom R.H. Whittaker Cell type, wall,
mode of nutrition,
motility
 TAXONOMY can be understand
by general example
Nomenclature
• Identification Providing a formal name
• Distinguishing features
genus & species
• Engine size
• Ford Crown Victoria
•Number of passengers • Toyota Camry
• Type of transmission Nomenclature • Honda Civic

Identifica
tion
• Classification
• Organization into groups
Classification
• Car
• Truck
• SUV
• Van (1701-1778)
TAXONOMY

*Kingdom/ Dunia : seluruh organisme dalam hierarki ini

*Phylum atau divisi : sekelompok kelas yang berkerabat
*Class/ Kelas : sekelompok ordo yang serupa
*Order/ Ordo : sekelompok famili yang serupa
*Family : sekelompok genus yang serupa
*Genus : sekelompok spesies yang serupa
*Species : sekelompok organisme berkerabat dekat

(1701-1778)
TAXONOMY

(1701-1778)
KLASIFIKASI secara Konvensional
• Bentuk sel
• Ukuran sel
• Morfologi koloni
• Karakteristik ultrastruktur
• Kebiasaan pewarnaan
• Mekanisme pergerakan
• Isi sel
• Sumber karbon & nitrogen
• Bahan penyusun dinding
sel
• Sumber energi
• Hasil Fermentasi
• Suhu pertumbuhan
optimum & kisarannya
• Toleransi osmosis
• Hubungan oksigen
• pH optimum & kisaran
pertumbuhan
• Sensitivitas terhadap
metabolic inhibitors &
antibiotics
KLASIFIKASI secara Phylogeny
 Menunjukkan hubungan evolusioner dan sejarah di
antara organisme

 Beberapa diperoleh dari data fosil

 Hanya mungkin dengan menggunakan teknik molekuler:

Genetic Homology:
Base composition (GC ratio)
Nucleic acid hybridisation.
Ribosomal RNA (rRNA) sequence analysis
Protein profiles and amino acid sequences
KLASIFIKASI secara Phylogeny
100 YA9 KU1401312 C03

55 betaproteobacteria Ralstonia mannitolilytica

YA9 KU1401312 E04

77 100 betaproteobacteria Burkholderia fungorum

YA9 KU1401312 H02

β-proteobacteria
100
betaproteobacteria Variovorax paradoxus
100
100 YA9 KU1401312 D05
100 betaproteobacteria Acidovorax delafieldii
YA9 KU1401312 B02
98 100 betaproteobacteria Alcaligenes aquatilis

100 YA10 KU140314 D08

gammaproteobacteria Klebsiella pneumoniae

100 YA9 KU1401312 H01

83
gammaproteobacteria Moraxella osloensis

100 YA10 KU140314 A08 γ-proteobacteria

53
gammaproteobacteria Acinetobacter oleivorans
65 YA9 KU1401312 F04
100 gammaproteobacteria Pseudomonas vancouverensis

100 YA9 KU1401312 G03

alphaproteobacteria Ochrobactrum pseudintermedium
α-proteobacteria
100 YA9 KU1401312 C04
actinobacteria Gordonia polyisoprenivorans

100 YA9 KU1401312 H05

100
actinobacteria Mycobacterium vanbaalenii
actinobacteridae
99 YA9 KU1401312 G05
99 actinobacteria Mycobacterium gilvum

0.02
Phylogenetic Tree
KLASIFIKASI secara Phylogeny

known
novel
Microbial Life, BOX 17.5
NOMENCLATURE

 Penamaan ilmiah dari suatu mikroorganisme

 Diatur oleh Peraturan Internasional (The International Code
of Nomenclature of Prokaryotes).
 Species/genus baru divalidasi dan dipublikasi di The
International Journal of Systematic and Evolutionary
Microbiology
(former: The International Journal of Bacteriology)

(1701-1778)
NOMENCLATURE
Peraturan untuk Penamaan Mikroorganisme

 Hanya ada 1 nama yang benar untuk 1 organisme

 Nama yang menyebabkan kesalahan harus ditolak
 Semua nama harus ditulis dalam huruf Latin
- Kata pertama (Genus) selalu ditulis dengan huruf besar
- Kata kedua (Species) tidak ditulis dengan huruf besar
- Nama genus dan species  Species, ditulis dengan
digarisbawahi atau dicetak miring
- Nama yang benar dari satu species atau taksa yang lebih
tinggi harus ditentukan oleh publikasi yang sah, dan
(1701-1778)
dan nanya harus sesuai dengan peraturan penamaan
NOMENCLATURE
 Mungkin merupakan suatu penghargaan bagi seorang ilmuwan

 Asal usul dalam bahasa Latin

Contoh. Escherichia coli (E. coli)
- penemu: Theodor Escherich
- menjelaskan tempat hidupnya (habitat) (colon/intestine)

Contoh. Staphylococcus aureus (S. aureus)

- Clustered (staphylo), spherical (cocci)
- Gold colored colonies (aureus)

Contoh. Ralstonia solanacearum (R. solanacearum)

- penemu : Ericka Ralston
- menginfeksi tanaman famili Solanaceae (1701-1778)
NOMENCLATURE

Nama umum/nama deskriptif :

Nama-nama mikroorganisme mungkin umum digunakan,

tapi bukan nama secara taksonomi
Contoh:
• Tubercle bacillus / disease = tuberculosis
(Mycobacterium tuberculosis)
• Meningococcus/ disease = meningitis
(Neiserria meningitidis)
• Group A streptococcus
(Streptococcus pyogenes)

(1701-1778)
Scientific Name

Scientific Binomial Source of Genus Source of

Name Specific Epithet
Klebsiella Honors Edwin Klebs The disease
pneumoniae
Pfiesteria piscicida Honors Lois Pfiester Disease in fish

Salmonella Honors Daniel Stupor (typh-) in

typhimurium Salmon mice (muri-)
Streptococcus Chains of cells Forms pus (pyo-)
pyogenes (strepto-)
Penicillium Tuftlike (penicill-) Produces a yellow
chrysogenum (chryso-) pigment
Trypanosoma cruzi Corkscrew-like Honors Oswaldo
(trypano-, borer; Cruz
soma-, body) (1701-1778)
IDENTIFIKASI

Bergey's Manual
- Metode untuk membedakan dan mengidentifikasi
bakteri yang disusun dalam Bergey's Manual of
Determinative Bacteriology

- Bergey's Manual of Systematic Bacteriology

Menyediakan penjelasan mengenai karakteristik fisik dan
kimia dan sistem identifikasi dari bakteri yang penting
IDENTIFIKASI
Bergey’s Manual of Systematic Bacteriology
• Morphological characteristics
• Presence of various enzymes
• Serological tests
• Phage typing
• Fatty acid profiles
• DNA finger printing (Genetic & molecular analysis)
• G + C base composition
• DNA analysis using genetic probes
• Nucleic acid sequencing & rRNA analysis
IDENTIFIKASI : METODE
• Morphological
characteristics:
Useful for
identifying
eukaryotes

• Differential
staining: Gram
staining, acid-fast
staining

• Biochemical tests:
Determines
presence of
bacterial enzymes A dichotomous key
IDENTIFIKASI
Dengan Metode Karakteristik Morfologi

Aspergillus E. coli

Streptomyces Penicillium
IDENTIFIKASI
Dengan Metode Karakteristik Morfologi

Kultur pada Agar Miring

IDENTIFIKASI BAKTERIA
Dengan Metode Klasik Mikrobiologi
IDENTIFIKASI BAKTERIA
Dengan Menggunakan Tes Biokimia
IDENTIFIKASI BAKTERIA
Dengan Menggunakan Tes Biokimia
IDENTIFIKASI

Design a rapid test for

a Staphylococcus aureus
 IDENTIFIKASI BAKTERIA
Dengan Menggunakan SEROLOGICAL TEST
Serology
(ilmu yang mempelajari tentang serum dan respon imun nya)

Combine known antiserum +

unknown bacterium
Slide agglutination
ELISA
Western blot
Southern Blot
DNA chip
IDENTIFIKASI BAKTERIA
Dengan Menggunakan PHAGE TYPING

Menentukan kepekaan suatu

strain terhadap phage atau
virus bakteri tertentu
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
IDENTIFIKASI BAKTERIA Dari Sample Tanah
Dengan Menggunakan DNA FINGERPRINRTING
-100 mL MM
-including 10 mg
pyrene
- 1 g soil sample
1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

Minggu I II III IV V VI VII

Enrichment culture
IDENTIFIKASI BAKTERIA Dari Sample Tanah
Dengan Menggunakan DNA FINGERPRINRTING
The flask was incubated under aerobic condition
The flask was shaken on an orbital shaker at room
temperature at speed of 120 rpm in the dark
condition.

Transfered culture to the solid medium which

containing pyrene.
The colonies show small and transparent
Pick up the colonies into the other plate

Isolated Bacteria

Pick up single colony for colony

PCR
IDENTIFIKASI BAKTERIA Dari Sample Tanah
Dengan Menggunakan DNA FINGERPRINRTING
PCR Amplification

PCR Cycler (Machine)

IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING

M 9 10 11 12 13 14 15 16 M

100 bp

PCR Result
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
PCR technology and Sequencing
Comparing
C G C C T T G A A G G C C G C C G T C T C G T G C C G G T C G T T C T G G C G G G T G C C C G AC C C G T G C G C G T T G AT G T A G T C G A T G T C C T C G G G G G C
140 150 160 170 180 190 200 210

with bank
data.
DNA extraction Sequencing
BLAST
Program

Library
Primer design
screening
and PCR

TA cloning
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
16S PCR technology DGGE (Denaturing Gradient Gel
TRFLP (Terminal Restriction Electrophoresis)
Fragment Length Polymorphism)

FISH (Fluorescent In Situ

SIP (Stable Isotop Probing) Hybridization
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
TRFLP (Terminal Restriction
Fragment Length Polymorphism)
* Mixed population is amplified
using a 16S primer with a
fluorescent tag

* PCR product is cut with a 4bp

cutting restriction endonuclease cut with 4bp RE
(RE)

* Different sequences will give

different length fragments

* Sample is injected into a

capillary sequencer to sort FU
fragments by size
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
TRFLP (Terminal Restriction
Fragment Length Polymorphism)
 Advantages

 Very sensitive

 Fast, easy and cheap

 Disadvantages

 Can NOT cut bands to get

sequence data

 Requires capillary sequencer

 Hard to distinguish noise from

little peaks sometimes
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
DGGE (Denaturing Gradient Gel Electrophoresis)
simple complex
 PCR products of mixed
communities are loaded on a gel
with a gradient of denaturant
20%
 Typically 20-80% formamide

 double stranded DNA will run down

the gel until it melts

 Melting determined by sequence

and GC content

 Different sequences migrate

different distances
80%
 You obtain a ‘barcode’ of the
community
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
DGGE (Denaturing Gradient Gel Electrophoresis)
 Advantages

 Can cut individual bands and

clone or sequence them

 Can detect very small

differences in DNA sequences

 Disadvantages

 High complexity samples give

smears

 Requires specialized gel rig

 Acryl-amide is highly toxic

IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
DGGE (Denaturing Gradient Gel Electrophoresis)

DGGE Apparatus
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
SIP (Stable isotope probing)
Bacterial population 13C apple pie

+  A population is grown on a substrate

that contains 13C carbon

grow on
labeled  Cells that eat the 13C labeled substrate
substrate will incorporate it into their DNA.
Dormant cells will not

12C DNA CsCl gradient  DNA extracted and heavy (13C

containing) DNA is separated from
light (only 12C containing) DNA by
extract 13C
DNA/RNA DNA CsCl density gradient centrifugation

 The heavy band is isolated and the

community analyzed by PCR – TA
cloning approach
centrifugation
 WS01ST2CH16

IDENTIFIKASI BAKTERIA Dengan

69
WS01ST3CH19
WS01ST3CH4
70
WS01ST2CH36
96
WS01ST5CH4
WS01ST7CH32
WS01ST7CH38
WS01ST6CH15
WS01ST6CH37
52 WS01ST6CH17
62 WS01ST6CH2
WS01ST7CH7
WS01ST6CH19
WS01ST7CH4

DNA FINGERPRINRTING
WS01ST5CH1
WS01ST6CH6
WS01ST2SY14
77 WS01ST2SY18
WS01ST6CH21
WS01ST5CH11
WS01ST6CH22
Diatoms
Cylindrotheca sp
WS01ST4CH12
WS01ST7CH1
WS01ST5CH14
WS01ST2SY17
Detonula confervacea
Odontella sinensis
WS01ST4CH15
WS01ST2CH2
WS01ST4CH20

SIP (Stable isotope probing)

95 WS01ST7CH27
WS01ST4CH36
55 WS01ST7CH18
WS01ST6CH25
WS01ST2CH4
WS01ST4CH31

Who is there ?
WS01ST7CH19
Skeletonema costatum
WS01ST4CH14
54 WS01ST6CH1
50 Phaeodactylum tricornutum
Bollidomonas pacifica
WS01ST7CH3 Bollidophytes
Bollidomonas mediterranea
WS01ST4CH16
Dictyochophyceae
76 Pseudopedinella elastica
100 WS01ST4CH4
67 WS01ST6CH33 Xanthophyceae
Heterococcus caespitosus
74 WS01ST1CH14
99 WS01ST3CH27
74 WS01ST8CH5
Eustigmatophytes
Nannochloropsis CCMP533
WS01ST1CH4
100 WS01ST1CH9
WS01ST3CH8
WS01ST1CH1
98
WS01ST3CH3
WS01ST1CH8
WS01ST3CH36
WS01ST8CH16
WS01ST1CH33
WS01ST8CH3
97 WS01ST8CH4
P994AH1
52
WS01ST8CH2
97 WS01ST8CH9
P994BH5
WS01ST1CH3
WS01ST6CH16
WS01ST5CH18
WS01ST1CH5
WS01ST2CH10 Prymnesiophytes
Chrysochromulina hirta
WS01ST1CH27
96 WS01ST3CH24
WS01ST4CH34
97 WS01ST7CH21
99 WS01ST8CH14
WS01ST8CH23
WS01ST8CH26
Emiliania huxleyi
Umbilicospaera sibogae
WS01ST5CH10
Chrysochromulina parva
WS01ST3CH23
Calcidiscus leptoporus
Platychrysis sp
Prymnesium parvum
P994AH12
WS01ST5CH2
99 Unk nown deeply rooted chromophytes
WS01ST8CH12
73 WS01ST8CH15

0.05

WS01ST3SY29
WS01ST7SY24
A13
WS01ST3SY2
P99SY12
S13
GG3L
P99SY5
B13
N5D
P99SY1 Marine Synechococcus
WS01ST2SY27
WS01ST6SY9
70 J15
WS01ST6SY3
WS01ST8SY9
WS01ST8SY18
WS01ST2SY30
70
WS01ST2SY26
95 WS01ST2SY4
98 P99SY22
Prochlorococcus marinus PAC1
WS01ST2SY19
WS01ST8SY4
WS01ST8SY26
77 Prochlorococcus marinus SB

Who is eating 91
83
Prochlorococcus marinus GP2

WS01ST3SY1
WS01ST3SY5
WS01ST2SY24
WS01ST2SY33
WS01ST2SY35
WS01ST1SY15
Prochlorococcus

apple pie ?
Hydrogenovibrio marinus
98 WS01ST4SY12
97 WS01ST8SY15 Trichodesmium
65
Trichodesmium thiebautii
Prochlorothrix hollandica
66 WS01ST4SY3
78 WS01ST6SY8
81
81
WS01ST5SY21
Pycnococcus provasolii
WS01ST3SY25
WS01ST1SY3
99 WS01ST8SY13
WS01ST8SY25
96 WS01ST8SY3
Spniach

Which microbial populations are active?

WS01ST1SY10
WS01ST8SY7
70 WS01ST5SY4
99 WS01ST7SY6
Chlamydomonas reinhardtii
74 WS01ST2SY2
WS01ST4SY17 Chlorophytes
97 WS01ST3SY26
87 WS01ST7SY29
78 WS01ST3SY4
WS01ST4SY39
81 WS01ST4SY7
Chlorella
Chlorella ellipsoidea

Don’t need to cultivate populations

69 Bathycoccus prasinos
WS01ST1SY35
Platydorina caudata
Yamagishiella unicocca
WS01ST5SY7
WS01ST4SY23
92 WS01ST6SY14
67 WS01ST5SY20
WS01ST6SY26
0.05
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
FISH (Fluorescent In Situ Hybridization)

 A cell population is fixed with

formaldehyde

 The cell membranes are

permeablized

 DNA or RNA probe is hybridized to

cells In-Situ i.e. while the cells are
still mostly intact

 The oligonucleotide contains a

fluorescent label, which can be
visualized by epifluorescence
microscopy
IDENTIFIKASI BAKTERIA
Dengan Menggunakan DNA FINGERPRINRTING
FISH (Fluorescent In Situ Hybridization)

 Advantages

 Allows visualization of a particular

population of cells (e.g. a species of
interest)
 Gives quantitative information about a
(bacterial population) microbial population
 Can probe for DNA, mRNA and
ribosomal RNA

 Disadvantages
 Cross-hybridization

 Different groups often do not add up to

100% of the population
 Relatively expensive and time consuming
(chromosome mapping)